Liver stromal cells for prevention and treatment of immune responses in transplantation

ABSTRACT

The present invention encompasses methods and compositions for reducing an immune response to a transplant in a recipient by treating said recipient with an amount of liver stromal cells effective to reduce or inhibit host rejection of the transplant. Also disclosed is a method of inducing a reduced immune response against a host by foreign tissue, i.e., graft versus host disease, by treatment with liver stromal cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. application Ser. No.11/222,316 filed Sep. 8, 2005, now pending; which claims the benefitunder 35 U.S.C. §119(e) to U.S. Application Ser. No. 60/609,077 filedSep. 10, 2004, now expired. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

BACKGROUND OF THE INVENTION

The liver is a dynamic organ that plays an important role in a varietyof physiological processes. The complex functions of the liver includemetabolism, storage, excretion, secretion of plasma proteins such asalbumin and detoxification of harmful substances by enzymes of thecytochrome P-450 system. In addition, the usually quiescent liver isalso capable of remarkable mitotic activities under certaincircumstances. The major cell population of the liver is the parenchymalcells (PC), also known as hepatocytes. The liver also contains severalother cell types such as endothelial cells, adipocytes, fibroblasticcells and Kupffer cells. The ability of liver cells to undergo rapidregeneration, when the liver is damaged or partially removed, makes theliver a potential source of stem cells.

It is currently believed that the liver has a stem cell and lineagesystem which has several parallels to the gut, skin and hemopoieticsystems (Sigal et al., 1993 Amer. J. Physiol., 263:139-148). As such,there are progenitor cell populations in the liver of animals of allages. These cells when isolated from the liver may serve as potentialcandidates for cell therapy.

The mammalian immune system plays a central role in protectingindividuals from infectious agents and preventing tumor growth. However,the same immune system can produce undesirable effects such as therejection of cell, tissue and organ transplants from unrelated donors.The immune system does not distinguish beneficial intruders, such as atransplanted tissue, from those that are harmful, and thus the immunesystem rejects transplanted tissues or organs. Rejection of transplantedorgans is generally mediated by alloreactive T cells present in the hostwhich recognize donor alloantigens or xenoantigens.

Immunologic tolerance is an actively induced unresponsiveness to aspecific antigen as the result of antigen-induced functionalinactivation or death of lymphocytes that are specific for that antigen.Antigens that induce such tolerance are termed “tolerogens,” so as to bedistinguished from immunogens which are antigens that generate immuneresponses. One mechanism of B cell tolerance and failure to produceantibodies involves the interaction of antigens with specific B cells(the first step in B cell activation) in the absence of stimulation byhelper T cells or other antigen presenting cells (the second step in Bcell activation). Other mechanisms of B cell tolerance have beenproposed. For example, B cells can become anergic due to a block insurface immunoglobulin-mediated signaling (“antigen-competition”), inthe absence of T cells. Additionally, in the absence of co-stimulationby an antigen presenting cell, strong crosslinking of B cell surfaceimmunoglobulins by an antigen can induce apoptotic death of normal,mature B cells, but may not induce apoptosis in B cells that produceautoimmune antibodies (Tsubata et al., 1994, Curr. Biol. 4:8-17).

T cell tolerance is achieved 1) in the thymus where thymocytes reactivefor self-peptides are eliminated by clonal deletion (central tolerance),and 2) in the periphery by exposure to self-antigens under tolerogenicconditions (peripheral tolerance). Clonal deletion can also result fromexpression of cell death molecules on antigen presenting cells. Classicexamples of cell death molecules are Fas ligand (FasL) and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL ligand), whichligate their receptors, Fas and DR4, respectively, on activated T cells,inducing apoptosis of the T cells. The interaction of CD27, a member ofthe TNFR superfamily, and the CD27-ligand (CD70) also induces T cellapoptosis.

The transplantation of cells, tissues, and organs between geneticallydisparate individuals invariably is associated with risk of graftrejection. Nearly all cells express products of the majorhistocompatibility complex, MHC class I molecules. Further, many celltypes can be induced to express MHC class II molecules when exposed toinflammatory cytokines. Additional immunogenic molecules include thosederived from minor histocompatibility antigens such as Y chromosomeantigens recognized by female recipients. Rejection of allografts ismediated primarily by T cells of both the CD4 and CD8 subclasses(Rosenberg et al., 1992 Annu. Rev. Immunol. 10:333). Alloreactive CD4 Tcells produce cytokines that exacerbate the cytolytic CD8 response toalloantigen. Within these subclasses, competing subpopulations of cellsdevelop after antigen stimulation that are characterized by thecytokines they produce. Th1 cells, which produce IL-2 and IFN-γ, areprimarily involved in allograft rejection (Mossmann et al., 1989 Annu.Rev. Immunol. 7:145). Th2 cells, which produce IL-4 and IL-10, candown-regulate Th1 responses through IL-10 (Fiorentino et al. 1989 J.Exp. Med. 170:2081). Indeed, much effort has been expended to divertundesirable Th1 responses toward the Th2 pathway. Undesirablealloreactive T cell responses in patients (allograft rejection, graftversus host disease) are typically treated with immunosuppressive drugssuch as prednisone, azathioprine, and cyclosporine A. Unfortunately,these drugs generally need to be administered for the life of thepatient and they have a multitude of dangerous side effects includinggeneralized immunosuppression.

A major goal in organ transplantation is the permanent engraftment ofthe donor organ without inducing a graft rejection immune responsegenerated by the recipient, while preserving the immunocompetence of therecipient against other foreign antigens. Typically, in order to preventhost rejection responses, nonspecific immunosuppressive agents such ascyclosporine, methotrexate, steroids and FK506 are used. These agentsmust be administered on a daily basis and if administration is stopped,graft rejection usually results. However, a major problem in usingnonspecific immunosuppressive agents is that they function bysuppressing all aspects of the immune response, thereby greatlyincreasing a recipient's susceptibility to infection and other diseases,including cancer.

Furthermore, despite the use of immunosuppressive agents, graftrejection still remains a major source of morbidity and mortality inhuman organ transplantation. Most human transplants fail within 10 yearswithout permanent graft acceptance. Only 50% of heart transplantssurvive 5 years and 20% of kidney transplants survive 10 years. (Opelzet al., 1981, Lancet 1:1223).

It is currently believed that a successful transplantation is dependenton the prevention and/or reduction of an unwanted immune response by ahost to a transplant mediated by immune effector cells to avert hostrejection of donor tissue. Also advantageous for a successfultransplantation is a method to eliminate or reduce an unwanted immuneresponse by a donor tissue against a recipient tissue known as graftversus host disease. Thus, there is long-felt need for methods tosuppress or otherwise prevent an unwanted immune response associatedwith transplantation of cells, tissues, and organs between geneticallydisparate individuals. The present invention meets this need.

SUMMARY OF THE INVENTION

The invention includes a method of reducing an immune response to atransplant in a recipient by treating the recipient with an amount ofliver stromal cells (LSCs) effective to reduce or inhibit host rejectionof the transplant. The invention also includes a method of inducing areduced immune response against a host by foreign tissue, i.e., graftversus host disease, by treatment with LSCs. The LSCs can beadministered before, at the same time as, or after the transplant.

The invention also includes a method of treating a transplant recipientto reduce in the recipient an immune response of effector cells againstan alloantigen to the effector cells comprising administering to atransplant recipient LSCs in an amount effective to reduce an immuneresponse of effector cells against an alloantigen to the effector cells,whereby in the transplant recipient the effector cells have a reducedimmune response against the alloantigen.

In one embodiment, the effector cell is a T cell.

In another embodiment, the T cell is from a donor and the alloantigen isfrom the recipient.

In another embodiment, the T cell is from a recipient and thealloantigen is from a donor.

In yet another embodiment, the T cell is present in the transplant.

In a further embodiment, the transplant is bone marrow.

In another embodiment, the transplant is a hematopoietic stem cell.

In one embodiment, the transplant is a neural stem cell.

In a further embodiment, the LSCs are expanded in culture prior toadministering to a transplant recipient.

In another embodiment, the effector cells are T cells from a donorpreviously activated by contacting the T cells with a cell or a tissuefrom the recipient prior to transplantation in order to activate the Tcells, and further wherein the immune response is the reactivation ofthe T cells.

In another embodiment, the LSCs are administered to the transplantrecipient to treat rejection of the transplant by the recipient.

In yet another embodiment, the LSCs are human LSCs.

In one embodiment, the method further comprises administering to therecipient an immunosuppressive agent.

In one embodiment, the transplant is a solid organ. Preferably, thesolid organ is selected from the group consisting of heart, pancreas,kidney, lung and liver.

In a further embodiment, the LSCs are administered intravenously to therecipient.

In another embodiment, the effector cells are cells of a recipient ofthe donor transplant.

In yet another embodiment, the LSCs are genetically modified.

The invention also includes a method for treating a transplant recipientto reduce in the recipient an immune response of effector cells againstan alloantigen to the effector cells comprising transplanting to atransplant recipient a transplant treated with LSCs in an amounteffective to reduce an immune response of effector cells against analloantigen to the effector cells, whereby in the transplant recipientthe effector cells have a reduced immune response against thealloantigen.

The invention also includes a method of reducing an immune response ofeffector cells against allogeneic cells comprising treating the effectorcells with LSCs.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiment(s) which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown.

FIG. 1 is a graph depicting the immunogenicity of liver stromal cells(LSCs).

FIG. 2 is a graph depicting suppression of the (mixed lymphocytereaction (MLR) response by fibroblast and stromal cells derived fromvarious sources.

FIG. 3 is a graph demonstrating that neural stem cells (NSCs) stimulateproliferation of allogeneic T cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that stromal cells fromliver (LSCs) possess novel immunological characteristics and thereforecan be useful in transplantation of a transplant, for example abiocompatible lattice or a donor tissue, organ or cell, by reducingand/or eliminating an immune response against the transplant by therecipient's own immune system. As described more fully below, LSCs playa role in inhibiting and/or preventing allograft rejection of atransplant.

In addition, the data disclosed herein also demonstrate that LSCs areuseful in the inhibition and/or prevention of an unwanted immuneresponse by a donor transplant, for example, a biocompatible lattice ora donor tissue, organ or cell, against a recipient tissue known as graftversus host disease.

Accordingly, the present invention encompasses methods and compositionsfor reducing and/or eliminating an immune response to a transplant in arecipient by treating the recipient with an amount of LSCs effective toreduce or inhibit host rejection of the transplant. Also encompassed aremethods and compositions for reducing and/or eliminating an immuneresponse in a host by the foreign transplant against the host, i.e.,graft versus host disease, by treating the donor transplant and/orrecipient of the transplant liver stromal cells in order to inhibit orreduce an adverse response by the donor transplant against therecipient.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

As used herein, the term “biocompatible lattice,” is meant to refer to asubstrate that can facilitate formation into three-dimensionalstructures conducive for tissue development. Thus, for example, cellscan be cultured or seeded onto such a biocompatible lattice, such as onethat includes extracellular matrix material, synthetic polymers,cytokines, growth factors, etc. The lattice can be molded into desiredshapes for facilitating the development of tissue types. Also, at leastat an early stage during culturing of the cells, the medium and/orsubstrate is supplemented with factors (e.g., growth factors, cytokines,extracellular matrix material, etc.) that facilitate the development ofappropriate tissue types and structures.

As used herein, the term “bone marrow stromal cells,” “stromal cells,”“mesenchymal stem cells” or “MSCs” are used interchangeably and refer tothe small fraction of cells in bone marrow which can serve as stemcell-like precursors to osteocytes, chondrocytes, monocytes, andadipocytes. Bone marrow stromal cells have been studied extensively(Castro-Malaspina et al., 1980, Blood 56:289-30125; Piersma et al.,1985, Exp. Hematol 13:237-243; Simmons et al., 1991, Blood 78:55-62;Beresford et al., 1992, J. Cell. Sci. 102:341-3 51; Liesveld et al.,1989, Blood 73:1794-1800; Liesveld et al., Exp. Hematol 19:63-70;Bennett et al., 1991, J. Cell. Sci. 99:131-139). Bone marrow stromalcells may be derived from any animal. In some embodiments, stromal cellsare derived from primates, preferably humans.

As used herein, “liver stromal cell” or “LSC” refers to a small fractionof fibroblastic-type cells derived from liver. LSCs, when contacted withT cells from an individual that is not the same individual from whichthe LSCs were obtained, do not elicit a T cell response. In addition,LSCs are able to suppress alloreactive T cell proliferation during animmune response. For example, LSCs can suppress a mixed lymphocytereaction (MLR) between allogeneic T cells and peripheral bloodmononuclear cells (PBMCs).

“Neural stem cell” or “NSC” is used herein to refer to undifferentiated,multipotent, self-renewing neural cell. A neural stem cell is aclonogenic multipotent stem cell which is able to divide and, underappropriate conditions, has self-renewal capability and can terminallydifferentiate into neurons, astrocytes, and oligodendrocytes. Hence, theneural stem cell is “multipotent” because stem cell progeny havemultiple differentiation pathways. A neural stem cell is capable ofself-maintenance, meaning that with each cell division, one daughtercell will also be, on average, a stem cell.

“Graft” refers to a cell, tissue, organ or otherwise any biologicalcompatible lattice for transplantation.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

“Transplant” refers to a biocompatible lattice or a donor tissue, organor cell, to be transplanted. An example of a transplant may include butis not limited to skin, bone marrow, and solid organs such as heart,pancreas, kidney, lung and liver.

As defined herein, an “allogeneic liver stromal cell (LSC)” is obtainedfrom a different individual of the same species as the recipient.

“Donor antigen” refers to an antigen expressed by the donor tissue to betransplanted into the recipient.

“Alloantigen” is an antigen that differs from an antigen expressed bythe recipient.

As used herein, an “effector cell” refers to a cell which mediates animmune response against an antigen. In the situation where a transplantis introduced into a recipient, the effector cells can be therecipient's own cells which elicits an immune response against anantigen present in the donor transplant. In another situation, theeffector cell can be part of the transplant, whereby the introduction ofthe transplant into a recipient results in the effector cells present inthe transplant eliciting an immune response against the recipient of thetransplant.

By the term “treating a transplant recipient to reduce in said recipientan immune response of effector cells against an alloantigen to theeffector cells,” as the phrase is used herein, is meant decreasing theendogenous immune response against the alloantigen in a recipient by anymethod, for example administering LSCs to a recipient, compared with theendogenous immune response in an otherwise identical animal which wasnot treated with LSCs. The decrease in endogenous immune response can beassessed using the methods disclosed herein or any other method forassessing endogenous immune response in an animal.

As used herein, the term “growth medium” is meant to refer to a culturemedium that promotes growth of cells. A growth medium may contain animalserum but this is not always a required component in that the growthmedium may also be serum free.

As used herein, the term “growth factor product” refers to a protein,peptide, mitogen, or other molecule having a growth, proliferative,differentiative, or trophic effect on a cell. For example, growth factorproducts useful in the treatment of CNS disorders include, but are notlimited to, nerve growth factor (NGF), brain-derived neurotrophic factor(BDNF), the neurotrophins (NT-3, NT-4/NT-5), ciliary neurotrophic factor(CNTF), amphiregulin, FGF-1, FGF-2, EGF, TGFα, TGFβs, PDGF, IGFs, andthe interleukins; IL-2, IL-12, IL-13.

“Immunophenotype” of a cell is used herein to refer to the phenotype ofa cell in terms of the surface protein profile of a cell.

An “isolated cell” refers to a cell which has been separated from othercomponents and/or cells which naturally accompany the isolated cell in atissue or mammal.

As used herein, the term “modulate” is meant to refer to any change inbiological state, i.e., increasing, decreasing, and the like.

As used herein, the term “non-immunogenic” is meant to refer to thediscovery that LSCs do not induce proliferation of T cells in an MLR.However, the term non-immunogenic should not be limited to the absenceof induction of T cell proliferation in an MLR, but rather should alsobe construed to apply to the absence of T cell proliferation in vivofollowing administration of LSCs to an animal.

“Proliferation” is used herein to refer to the reproduction ormultiplication of cells. That is, proliferation encompasses productionof a greater number of cells, and can be measured by, among otherthings, simply counting the numbers of cells, measuring incorporation of³H-thymidine into the cell, and the like.

The term “stromal cell medium” as used herein, refers to a medium usefulfor culturing LSCs. A non-limiting example of a stromal cell medium is amedium comprising DMEM/F 12 Ham's, 10% fetal bovine serum, 100 Upenicillin/100 μg streptomycin/0.25 μg Fungizone. Typically, the stromalcell medium comprises a base medium, serum and anantibiotic/antimycotic. However, LSCs can be cultured in a stromal cellmedium without an antibiotic/antimycotic and supplemented with at leastone growth factor. The preferred base medium is DMEM/F 12 (1:1). Thepreferred serum is fetal bovine serum (FBS) but other sera may be usedincluding horse serum or human serum. Preferably up to 20% FBS is addedto the above media in order to support the growth of stromal cells.However, a defined medium can be used if the necessary growth factors,cytokines, and hormones in FBS for stromal cell growth are identifiedand provided at appropriate concentrations in the growth medium. It isfurther recognized that additional components may be added to theculture medium. Such components include but are not limited toantibiotics, antimycotics, albumin, growth factors, amino acids, andother components known in the art for the culture of cells. Antibioticswhich can be added into the medium include, but are not limited to,penicillin and streptomycin. The concentration of penicillin in theculture medium is about 10 to about 200 units per ml. The concentrationof streptomycin in the culture medium is about 10 to about 200 μg/ml.However, the invention should in no way be construed to be limited toany one medium for culturing stromal cells. Rather, any media capable ofsupporting stromal cells in tissue culture may be used.

As used herein, a “therapeutically effective amount” is the amount ofLSCs which is sufficient to provide a beneficial effect to the subjectto which the LSCs are administered.

As used herein “endogenous” refers to any material from or producedinside an organism, cell or system.

“Exogenous” refers to any material introduced from or produced outsidean organism, cell, or system.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, poly lysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those, known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

The term “genetic modification” as used herein refers to the stable ortransient alteration of the genotype of an LSC by intentionalintroduction of exogenous DNA. Preferably, the exogenous DNA is anisolated nucleic acid. The DNA may be synthetic, or naturally derived,and may contain genes, portions of genes, or other useful DNA sequences.The term “genetic modification” as used herein is not meant to includenaturally occurring alterations such as that which occurs throughnatural viral activity, natural genetic recombination, or the like.

Description

The present invention relates to the discovery that when liver stromalcells (LSCs) are contacted with T cells obtained from a differentindividual (allogeneic T cells), the allogeneic T cells do notproliferate. Prior art dogma suggests that when T cells are mixed withany other cell, T cell proliferation ensues. This phenomenon is known asa mixed lymphocyte reaction (MLR). The data disclosed herein demonstratethat T cells derived from an individual are not responsive to LSCsobtained from a different individual. Therefore, based upon thedisclosure herein, LSCs are not immunogenic to the immune system withrespect to manifesting a T cell response.

In addition to the non-immunogenic phenotype of LSCs with respect to Tlymphocytes in a different individual, the present invention alsorelates to the novel discovery that LSCs can suppress an MLR betweenallogeneic cells, for example between T cells from an individual andperipheral blood mononuclear cells (PBMCs) from another individual.These unexpected results demonstrate that LSCs can actively reduce theallogeneic T cell response in MLRs between T cells and PBMCs fromdifferent individuals. Moreover, as discussed in more detail elsewhereherein, this reduction is observed to occur in a dose dependent manner.This shows that LSCs can be used as a therapy to inhibit host rejectionof a transplant, and in addition, prevent or otherwise inhibit graftversus host disease following transplantation.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that the ability of LSCs to suppress an allogeneic Tcell response is not limited to an MLR between T cells and PBMCs fromdisparate individuals, but rather, the LSCs can be exploited to includesuppression of an MLR between T cells and any type of cell from adifferent individual, for example a neural stem cell (NSC), a livercell, a cardiac cell, a chondrocyte, a kidney cell, an adipose cell andthe like.

Accordingly, the present invention encompasses methods for reducingand/or eliminating an immune response to a transplant in a recipient byadministering to the recipient of the transplant an amount of LSCseffective to reduce or inhibit host rejection of the transplant. Withoutwishing to be bound to any particular theory, the LSCs that areadministered to the recipient of the transplant inhibit the activationand proliferation of the recipient's T cells.

I. Isolation and Culturing of LSCs

The LSCs useful in the methods of the present invention may be isolatedusing a variety of methods known to those skilled in the art. In apreferred method, an LSC is isolated from a mammalian subject,preferably a human subject.

Based upon the disclosure provided herein, LSCs can be obtained from anysource, for example, from the tissue donor, the transplant recipient oran otherwise unrelated source (a different individual or speciesaltogether). The LSCs may be autologous with respect to the T cells(obtained from the same host) or allogeneic with to the T cells. In thecase where the LSCs are allogeneic, the LSCs may be autologous withrespect to the transplant to which the T cells are responding to, or theLSCs may be obtained from an individual that is allogeneic with respectto both the source of the T cells and the source of the transplant towhich the T cells are responding to. In addition, the LSCs may bexenogeneic to the T cells (obtained from an animal of a differentspecies), for example rat LSCs may be used to suppress activation andproliferation of human T cells in MLRs.

In a further embodiment, LSCs used in the present invention can beisolated, from liver of any species of mammals, including but notlimited to, a human, a mouse, a rat, an ape, a gibbon, a bovine, and thelike. Preferably, the LSCs are isolated from a mouse or a rat. Morepreferably, the LSCs are isolated from a human.

Based upon the present disclosure, LSCs can be isolated and expanded inculture, i.e., in vitro, to obtain sufficient numbers of cells for theuse in the methods described herein. For example, LSCs can be isolatedfrom a human liver and cultured in complete medium (DMEM low glucosecontaining 4 mM L-glutamine, 10% FBS, and 1% Penicillin/Streptomycin).However, the invention should in no way be construed to be limited toany one method of isolating and culturing LSCs. Rather, any method ofisolating and culturing LSCs should be construed to be included in thepresent invention.

Any medium capable of supporting fibroblasts in cell culture may be usedto culture LSCs. Media formulations that support the growth offibroblasts include, but are not limited to, Minimum Essential MediumEagle, ADC-1, LPM (bovine serum albumin-free), F10 (HAM), F12 (HAM),DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-JacksonModification), Basal Medium Eagle (BME-with the addition of Earle's saltbase), Dulbecco's Modified Eagle Medium (DMEM-without serum), Yamane,IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15Medium, McCoy's 5A Medium, Medium M199 (M199E-with Earle's salt base),Medium M199 (M199H-with Hank's salt base), Minimum Essential MediumEagle (MEM-E-with Earle's salt base), Minimum Essential Medium Eagle(MEM-H-with Hank's salt base) and Minimum Essential Medium Eagle(MEM-NAA with non-essential amino acids), and the like. A preferredmedium for culturing LSCs is DMEM.

Any medium capable of supporting LSCs in vitro may be used to culturethe LSCs. Media formulations that can support the growth of LSCsinclude, but are not limited to, Dulbecco's Modified Eagle's Medium(DMEM), alpha modified Minimal Essential Medium (aMEM), and Roswell ParkMemorial Institute Media 1640 (RPMI Media 1640) and the like. Typically,0 to 20% Fetal Bovine Serum (FBS) or 1-20% horse serum is added to theabove medium in order to support the growth of LSCs. However, a definedmedium can also be used if the growth factors, cytokines, and hormonesnecessary for culturing LSCs are provided at appropriate concentrationsin the growth medium. Media useful in the methods of the invention maycontain one or more compounds of interest, including but not limited toantibiotics, mitogenic or differentiation compounds useful for theculturing of LSCs. The cells may be grown at temperatures between 27° C.to 40° C., preferably 31° C. to 37° C., and more preferably in ahumidified incubator. The carbon dioxide content may be maintainedbetween 2% to 10% and the oxygen content between 1% and 22%. However,the invention should in no way be construed to be limited to any onemethod of isolating and culturing LSCs. Rather, any method of isolatingand culturing LSCs should be construed to be included in the presentinvention.

Additional non-limiting examples of media useful in the methods of theinvention contain fetal serum of bovine or other species at aconcentration at least 1% to about 30%, preferably at least about 5% to15%, most preferably about 10%. Embryonic extract of chicken or otherspecies can be present at a concentration of about 1% to 30%, preferablyat least about 5% to 15%, most preferably about 10%.

Antibiotics which can be added into the medium include, but are notlimited to, penicillin and streptomycin. The concentration of penicillinin the culture medium is about 10 to about 200 units per ml. Theconcentration of streptomycin in the culture medium is about 10 to about200 μg/ml.

Following isolation, LSCs are incubated in culture medium in a cultureapparatus for a period of time, or until the cells reach confluency,before passing the cells to another culture apparatus. Following theinitial plating, the cells can be maintained in culture for a period ofabout 6 days to yield the Passage 0 (P0) population. The cells can bepassaged for an indefinite number of times, each passage comprisingculturing the cells for about 6-7 days, during which the cell doublingtimes can range between 3-5 days. The culturing apparatus can be of anyculture apparatus commonly used in culturing cells in vitro. A preferredculture apparatus is a culture flask, and a more preferred cultureapparatus is a T-225 culture flask.

LSCs can be cultured in stromal cell medium for a period of time oruntil the cells reach a certain level of confluence. Preferably, thelevel of confluence is greater than 70%. More preferably, the level ofconfluence is greater than 90%. A period of time can be any timesuitable for the culture of cells in vitro. Stromal cell medium may bereplaced during the culturing of the LSCs at any time. Preferably, thestromal cell medium is replaced every 3 to 4 days. LSCs are thenharvested from the culture apparatus whereupon the LSCs can be usedimmediately or they can be cryopreserved and stored for use at a latertime. LSCs may be harvested by trypsinization, EDTA treatment, or anyother procedure used to harvest cells from a culture apparatus.

LSCs described herein may be cryopreserved according to routineprocedures. Preferably, about one to ten million cells are cryopreservedin stromal cell medium containing 10% DMSO in vapor phase of liquid N₂.Frozen cells can be thawed by swirling in a 37° C. bath, resuspended infresh growth medium, and grown as usual.

II. Therapy

As encompassed in the present invention, LSCs are typically isolatedfrom a human. If the cell of the present invention is to be transplantedinto a human subject, it is preferable that the LSC be isolated fromthat same subject so as to provide for an autologous transplant.However, allogeneic transplants are also contemplated by the presentinvention.

Thus, in another aspect of the invention, the administered LSCs may beallogeneic with respect to the recipient. An allogeneic LSC cell can beisolated from a donor that is a different individual of the same speciesas the recipient. Following isolation, the cell is cultured using themethods disclosed herein to produce an allogeneic product. The inventionalso encompasses an LSC that is xenogeneic with respect to therecipient.

Another embodiment of present invention encompasses the route ofadministering LSCs to the recipient of the transplant. LSCs can beadministered by a route which is suitable for the placement of thetransplant, i.e., a biocompatible lattice or a donor tissue, organ orcell, to be transplanted. LSCs can be administered systemically, i.e.,parenterally, by intravenous injection or can be targeted to aparticular tissue or organ, such as bone marrow. LSCs can beadministered via a subcutaneous implantation of cells or by injection ofthe cells into connective tissue, for example muscle.

LSCs can be suspended in an appropriate diluent, at a concentration offrom about 0.01 to about 5×10⁶ cells/ml. Suitable excipients forinjection solutions are those that are biologically and physiologicallycompatible with the LSCs and with the recipient, such as buffered salinesolution or other suitable excipients. The composition foradministration can be formulated, produced and stored according tostandard methods complying with proper sterility and stability.

The dosage of the LSCs varies within wide limits and may be adjusted tothe individual requirements in each particular case. The number of cellsused depends on the weight and condition of the recipient, the numberand/or frequency of administrations, and other variables known to thoseof skill in the art.

Between about 10⁵ and about 10¹³ LSCs per 100 kg body weight can beadministered to the individual. In some embodiments, between about1.5×10⁶ and about 1.5×10¹² cells are administered per 100 kg bodyweight. In some embodiments, between about 1×10⁹ and about 5×10¹¹ cellsare administered per 100 kg body weight. In some embodiments, betweenabout 4×10⁹ and about 2×10¹¹ cells are administered per 100 kg bodyweight. In some embodiments, between about 5×10⁸ cells and about 1×10¹cells are administered per 100 kg body weight.

III. Host Rejection

In another embodiment of the present invention, LSCs are administered tothe recipient prior to, or contemporaneously with a transplant to reduceand/or eliminate host rejection of the transplant. While not wishing tobe bound to any particular theory, LSCs can be used to condition arecipient's immune system to the transplant by administering LSCs to therecipient, prior to, or at the same time as transplantation of thetransplant, in an amount effective to reduce, inhibit or eliminate animmune response against the transplant by the recipient's T cells. TheLSCs affect the T cells of the recipient such that the T cell responseis reduced, inhibited or eliminated when presented with the transplant.Thus, host rejection of the transplant may be avoided, or the severitythereof reduced, by administering LSCs to the recipient, prior to, or atthe same time as transplantation.

In yet another embodiment, LSCs can be administered to the recipient ofthe transplant after the administration of the transplant. Further, thepresent invention comprises a method of treating a patient who isundergoing an adverse immune response to a transplant by administeringLSCs to the patient in an amount effective to reduce, inhibit oreliminate the immune response to the transplant, also known as hostrejection of the transplant.

The invention is based on the discovery that LSCs do not stimulateallogeneic T cell proliferation. As such, the invention encompassesusing LSCs to suppress T cell proliferation in response to transplant ofan exogenous organ, tissue or cells. The invention also includes amethod of administering an LSC to a patient in an amount effective toreduce an immune response with respect to T cell proliferation.

One skilled in the art would appreciate, based upon the disclosureprovided herein, that LSCs can be exploited to include suppression of Tcell proliferation in response to any type of organ, tissue or celltransplanted into a mammal and obtained from a different individual. Forexample, T cell proliferation in response to a cell including, but notlimited to a neural stem cell (NSC), a liver cell, a cardiac cell, achondrocyte, a kidney cell, an adipose cell, and the like, can besuppressed using LSCs.

The present invention encompasses a method of reducing and/oreliminating an immune response to a transplant in a recipient byadministering to the recipient of the transplant an amount of LSCseffective to reduce or inhibit host rejection of the transplant. Withoutwishing to be bound to any particular theory, the LSCs that areadministered to the recipient of the transplant inhibit the activationand proliferation of the recipient's T cells.

The transplant includes a biocompatible lattice or a donor tissue, organor cell, to be transplanted. An example of a transplant may include, butis not limited to stem cells, skin cells or tissue, bone marrow, neuralstem cells, and solid organs such as heart, pancreas, kidney, lung andliver.

IV. Graft Versus Host Disease

In addition to methods to reduce and/or eliminate host rejection of thetransplant, the present invention also provides a method of reducingand/or eliminating an immune response by a donor transplant against arecipient thereof (i.e., graft versus host reaction). Accordingly, thepresent invention encompasses a method of contacting a donor transplant,for example a biocompatible lattice or a donor tissue, organ or cell,with LSCs prior to transplantation of the transplant into a recipient.The LSCs serve to ameliorate, inhibit or reduce an adverse response bythe donor transplant against the recipient.

The invention is based on the discovery that LSCs do not stimulateallogeneic T cell proliferation. Based on the disclosure presentedherein, LSCs can suppress T cell proliferation in an MLR reaction. Theinvention also includes a method of administering an LSC to a mammal inan amount effective to reduce an immune response with respect to T cellproliferation.

As discussed elsewhere herein, LSCs can be obtained from any source, forexample, from the tissue donor, the transplant recipient or an otherwiseunrelated source (a different individual or species altogether) for theuse of eliminating or reducing an unwanted immune response by atransplant against a recipient of the transplant. Accordingly, LSCs canbe autologous, allogeneic or xenogeneic to the tissue donor, thetransplant recipient or an otherwise unrelated source.

In an embodiment of the present invention, the transplant is exposed toLSCs prior to transplantation of the transplant into the recipient. Inthis situation, an immune response against the transplant caused by anyalloreactive recipient cells would be suppressed by the LSCs present inthe transplant. The LSCs are allogeneic to the recipient and may bederived from the donor or from a source other than the donor orrecipient. In some cases, LSCs autologous to the recipient may be usedto suppress an immune response against the transplant. In another case,the LSCs may be xenogeneic to the recipient, for example mouse or ratLSCs can be used to suppress an immune response in a human. However, itis preferable to use human LSCs in the present invention.

In another embodiment of the present invention, the donor transplant canbe “preconditioned” or “pretreated” by treating the transplant prior totransplantation into the recipient in order to reduce the immunogenicityof the transplant against the recipient, thereby reducing and/orpreventing graft versus host disease. The transplant can be contactedwith cells or a tissue from the recipient prior to transplantation inorder to activate T cells that may be associated with the transplant.Following the treatment of the transplant with cells or a tissue fromthe recipient, the cells or tissue may be removed from the transplant.The treated transplant is then further contacted with LSCs in order toreduce, inhibit or eliminate the activity of the T cells that wereactivated by the treatment of the cells or tissue from the recipient.Following this treatment of the transplant with LSCs, the LSCs may beremoved from the transplant prior to transplantation into the recipient.However, some LSCs may adhere to the transplant, and therefore, may beintroduced to the recipient with the transplant. In this situation, theLSCs introduced into the recipient can suppress an immune responseagainst the recipient caused by any cell associated with the transplant.Without wishing to be bound to any particular theory, the treatment ofthe transplant with LSCs prior to transplantation of the transplant intothe recipient serves to reduce, inhibit or eliminate the activity of theactivated T cells, thereby preventing restimulation, or inducinghyporesponsiveness of the T cells to subsequent antigenic stimulationfrom a tissue and/or cells from the recipient. One skilled in the artwould understand based upon the present disclosure, that preconditioningor pretreatment of the transplant prior to transplantation may reduce oreliminate the graft versus host response.

For example, in the context of bone marrow or peripheral blood stem cell(hematopoietic stem cell) transplantation, attack of the host by thegraft can be reduced, inhibited or eliminated by preconditioning thedonor marrow by using the pretreatment methods disclosed herein in orderto reduce the immunogenicity of the graft against the recipient. Asdescribed elsewhere herein, a donor marrow can be pretreated with LSCsfrom any source, preferably with recipient LSCs in vitro prior to thetransplantation of the donor marrow into the recipient. In a preferredembodiment, the donor marrow is first exposed to recipient tissue orcells and then treated with LSCs. Although not wishing to be bound toany particular theory, it is believed that the initial contact of thedonor marrow with recipient tissue or cells function to activate the Tcells in the donor marrow. Treatment of the donor marrow with the LSCsinduces hyporesponsiveness or prevents restimulation of T cells tosubsequent antigenic stimulation, thereby reducing, inhibiting oreliminating an adverse effect induced by the donor marrow on therecipient.

In an embodiment of the present invention, a transplant recipientsuffering from graft versus host disease may be treated by administeringLSCs to the recipient to reduce, inhibit or eliminate the severitythereof from the graft versus host disease where the LSCs areadministered in an amount effective to reduce or eliminate graft versushost disease.

In this embodiment of the invention, preferably, the recipient's LSCsmay be obtained from the recipient prior to the transplantation and maybe stored and/or expanded in culture to provide a reserve of LSCs insufficient amounts for treating an ongoing graft versus host reaction.However, as discussed elsewhere herein, LSCs can be obtained from anysource, for example, from the tissue donor, the transplant recipient oran otherwise unrelated source (a different individual or speciesaltogether).

V. Advantages of Using LSCs

Based upon the disclosure herein, it is envisioned that the LSCs of thepresent invention can be used in conjunction with current modes, forexample the use of immunosuppressive drug therapy, for the treatment ofhost rejection to the donor tissue or graft versus host disease. Anadvantage of using LSCs in conjunction with immunosuppressive drugs intransplantation is that by using the methods of the present invention toameliorate the severity of the immune response in a transplantrecipient, the amount of immunosuppressive drug therapy used and/or thefrequency of administration of immunosuppressive drug therapy can bereduced. A benefit of reducing the use of immunosuppressive drug therapyis the alleviation of general immune suppression and unwanted sideeffects associated with immunosuppressive drug therapy.

It is also contemplated that the cells of the present invention may beadministered into a recipient as a “one-time” therapy for the treatmentof host rejection of donor tissue or graft versus host disease. Aone-time administration of LSCs into the recipient of the transplanteliminates the need for chronic immunosuppressive drug therapy. However,if desired, multiple administrations of LSCs may also be employed.

The invention described herein also encompasses a method of preventingor treating transplant rejection and/or graft versus host disease byadministering LSCs in a prophylactic or therapeutically effective amountfor the prevention, treatment or amelioration of host rejection of thetransplant and/or graft versus host disease. Based upon the presentdisclosure, a therapeutic effective amount of LSCs is an amount thatinhibits or decreases the number of activated T cells, when comparedwith the number of activated T cells in the absence of theadministration of LSCs. In the situation of host rejection of thetransplant, an effective amount of LSCs is an amount that inhibits ordecreases the number of activated T cells in the recipient of thetransplant when compared with the number of activated T cells in therecipient prior to administration of the LSCs. In the case of graftversus host disease, an effective amount of LSCs is an amount thatinhibits or decreases the number of activated T cells present in thetransplant.

An effective amount of LSCs can be determined by comparing the number ofactivated T cells in a recipient or in a transplant prior to theadministration of LSCs thereto, with the number of activated T cellspresent in the recipient or transplant following the administration ofLSCs thereto. A decrease, or the absence of an increase, in the numberof activated T cells in the recipient of the transplant or in thetransplant itself that is associated with the administration of LSCsthereto, indicates that the number of LSCs administered is a therapeuticeffective amount of LSCs.

Genetic Modification

In yet another embodiment of the present invention, the LSCs can be usedas a gene therapy vehicle for the expression of an exogenous gene in amammal. The benefit of using LSCs as a vehicle for gene therapy over thepresently used cells is based on the novel discovery that LSCs cansurvive for longer periods of time when compared with cells presentlyused in the art for gene therapy applications.

Unless otherwise stated, genetic manipulations are performed asdescribed in Sambrook et al. (2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York). The present inventionencompasses genetically modified LSCs, which have been engineered toexpress an exogenous gene. The exogenous gene can, for example, be anexogenous version of an endogenous gene (for example, a wild typeversion of the same gene can be used to replace a defective allelecomprising a mutation). The exogenous gene is usually but notnecessarily covalently linked with (i.e., “fused with”) one or moreadditional genes. Exemplary “additional” genes include a gene useful for“positive” selection to select cells that have incorporated theexogenous gene, and a gene useful for “negative” selection to selectcells that have incorporated the exogenous gene into the samechromosomal locus as the endogenous gene or both.

The cells of the present invention can also be used to express a foreignprotein or molecule for a therapeutic purpose or in a method of trackingtheir assimilation and/or differentiation in the recipient. Thus, theinvention encompasses expression vectors and methods for theintroduction of an isolated nucleic acid into LSCs with concomitantexpression of the isolated nucleic acid in the LSCs. Methods forintroducing and expressing DNA in a cell are well known to the skilledartisan and include those described, for example, in Sambrook et al.(2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York), and in Ausubel et al. (1997, Current Protocols inMolecular Biology, John Wiley & Sons, New York).

The isolated nucleic acid can encode a molecule used to track themigration, assimilation, and survival of LSCs once they are introducedin the recipient. Proteins useful for tracking a cell include, but arenot limited to, green fluorescent protein (GFP), any of the otherfluorescent proteins (e.g., enhanced green, cyan, yellow, blue and redfluorescent proteins; Clontech, Palo Alto, Calif.), or other tagproteins (e.g., LacZ, FLAG-tag, Myc, His₆, and the like).

Tracking the migration and assimilation of an LSC of the presentinvention is not limited to the use of a detectable molecule expressedby a vector or virus. The migration and assimilation of a cell can alsobe assessed using a series of probes that facilitate localization oftransplanted LSCs within a mammal. Tracking an LSC transplant mayfurther be accomplished using antibodies or nucleic acid probes forcell-specific markers associated with LSCs.

An isolated nucleic acid may be introduced to an LSC using viral vectors(retrovirus, modified herpes viral, herpes-viral, adenovirus,adeno-associated virus, lentiviral, and the like) or by direct DNAtransfection (lipofection, calcium phosphate transfection, DEAE-dextran,electroporation, and the like).

When the purpose of genetic modification of the cell is for theproduction of a biologically active substance, the substance willgenerally be one that is useful for the treatment of a given disorder.For example, it may be desired to genetically modify cells so that theysecrete a certain growth factor product.

The cells of the present invention can be genetically modified byintroducing an isolated nucleic acid into the cells, to produce amolecule such as a trophic factor, a growth factor, a cytokine, and thelike, which is beneficial to culturing the cells. In addition, thegenetically modified cells can provide an additional therapeutic effectto the patient when transplanted into a patient in need thereof.

Cells can also be modified to express a certain growth factor receptor(r) including, but not limited to, p75 low affinity NGFr, CNTFr, the trkfamily of neurotrophin receptors (trk, trkB, trkC), EGFr, FGFr, andamphiregulin receptors. Cells can be engineered to produce variousneurotransmitters or their receptors such as serotonin, L-dopa,dopamine, norepinephrine, epinephrine, tachykinin, substance-P,endorphin, enkephalin, histamine, N-methyl D-aspartate, glycine,glutamate, GABA, ACh, and the like. Useful neurotransmitter-synthesizinggenes include TH, dopa-decarboxylase (DDC), DBH, PNMT, GAD, tryptophanhydroxylase, ChAT, and histidine decarboxylase. Genes that encodevarious neuropeptides which may prove useful in the treatment of CNSdisorders, include substance-P, neuropeptide-Y, enkephalin, vasopressin,VIP, glucagon, bombesin, cholecystokinin (CCK), somatostatin, calcitoningene-related peptide, and the like.

The cells of the present invention can also be modified to express acytokine. The cytokine is preferably, but not exclusively selected fromthe group consisting of IL-12, TNFα, IFNα, IFNβ, IFNγ, IL-7, IL-2, IL-6,IL-15, IL-21, and IL-23.

According to the present invention, gene constructs which comprisenucleotide sequences that encode heterologous proteins are introducedinto the LSCs. That is, the cells are genetically altered to introduce agene whose expression has therapeutic effect in the individual.According to some aspects of the invention, LSCs from the individual tobe treated or from another individual, or from a non-human animal, maybe genetically altered to replace a defective gene and/or to introduce agene whose expression has therapeutic effect in the individual beingtreated.

In all cases in which a gene construct is transfected into a cell, theheterologous gene is operably linked to regulatory sequences required toachieve expression of the gene in the cell. Such regulatory sequencestypically include a promoter and a polyadenylation signal.

The gene construct is preferably provided as an expression vector thatincludes the coding sequence for a heterologous protein operably linkedto essential regulatory sequences such that when the vector istransfected into the cell, the coding sequence will be expressed by thecell. The coding sequence is operably linked to the regulatory elementsnecessary for expression of that sequence in the cells. The nucleotidesequence that encodes the protein may be cDNA, genomic DNA, synthesizedDNA or a hybrid thereof or an RNA molecule such as mRNA.

The gene construct includes the nucleotide sequence encoding thebeneficial protein operably linked to the regulatory elements and mayremain present in the cell as a functioning cytoplasmic molecule, afunctioning episomal molecule or it may integrate into the cell'schromosomal DNA. An isolated nucleic acid may be introduced into cellswhere it remains as separate genetic material in the form of a plasmid.Alternatively, linear DNA which can integrate into the chromosome may beintroduced into the cell. When introducing DNA into the cell, reagentswhich promote DNA integration into chromosomes may be added. DNAsequences which are useful to promote integration may also be includedin the DNA molecule. Alternatively, RNA may be introduced into the cell.

The regulatory elements for gene expression include: a promoter, aninitiation codon, a stop codon, and a polyadenylation signal. It ispreferred that these elements be operable in the cells of the presentinvention. Moreover, it is preferred that these elements be operablylinked to the nucleotide sequence that encodes the protein such that thenucleotide sequence can be expressed in the cells and thus the proteincan be produced. Initiation codons and stop codons are generallyconsidered to be part of a nucleotide sequence that encodes the protein.However, it is preferred that these elements are functional in thecells. Similarly, promoters and polyadenylation signals used must befunctional within the cells of the present invention. Examples ofpromoters useful to practice the present invention include but are notlimited to promoters that are active in many cells such as thecytomegalovirus promoter, SV40 promoters and retroviral promoters. Otherexamples of promoters useful to practice the present invention includebut are not limited to tissue-specific promoters, i.e., promoters thatfunction in some tissues but not in others; also, promoters of genesnormally expressed in the cells with or without specific or generalenhancer sequences. In some embodiments, promoters are used whichconstitutively express genes in the cells with or without enhancersequences. Enhancer sequences are provided in such embodiments whenappropriate or desirable.

The cells of the present invention can be transfected using well knowntechniques readily available to those having ordinary skill in the art.An isolated nucleic acid may be introduced into the cells using standardmethods where the cell expresses the protein encoded by the gene. Insome embodiments, cells are transfected by calcium phosphateprecipitation transfection, DEAE dextran transfection, electroporation,microinjection, liposome-mediated transfer, chemical-mediated transfer,ligand mediated transfer or recombinant viral vector transfer.

In some embodiments, recombinant adenovirus vectors are used tointroduce DNA with desired sequences into the cell. In some embodiments,recombinant retrovirus vectors are used to introduce DNA with desiredsequences into the cells. In some embodiments, standard CaP04, DEAEdextran or lipid carrier mediated transfection techniques are employedto incorporate desired DNA into dividing cells. Standard antibioticresistance selection techniques can be used to identify and selecttransfected cells. In other embodiments, DNA is introduced directly intocells by microinjection. Similarly, well-known electroporation orparticle bombardment techniques can be used to introduce foreign DNAinto the cells. A second gene is usually co-transfected or linked to thetherapeutic gene. The second gene is frequently a selectableantibiotic-resistance gene. Transfected cells can be selected by growingthe cells in an antibiotic that will kill cells that do not take up theselectable gene. In most cases where the two genes are unlinked andco-transfected, the cells that survive the antibiotic treatment haveboth genes in them and express both of them.

It should be understood that the methods described herein may be carriedout in a number of ways and with various modifications and permutationsthereof that are well known in the art. It may also be appreciated thatany theories set forth as to modes of action or interactions betweencell types should not be construed as limiting this invention in anymanner, but are presented such that the methods of the invention can bemore fully understood.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES Example 1 Characterization of Liver Stromal Cells Production ofLSCS

Adult cadaveric human livers from different donors designated Hu027 andHu029 were used. The liver was perfused via portal vein and hepaticartery with EGTA-containing buffer for 15 minutes and 0.06% collagenase(Sigma Chemical Company, St. Louis, Mo.) for 30 minutes at 34° C. Thecells were then passed through 1000, 500, 250 and 150 μm filters. Viablecells were fractionated under 500×g with COBE cell processor (GambroBCT, Lakewood, Colo.) using 2-step (9% and 17%; Hu027 and Hu029 cells)or 12.5% (H0107 cells) OptiPrep gradients (Axis-Shield PoC AS, Oslo,Norway). Cells were frozen in medium containing 80% Hypothermosol (BioLife Solutions Inc., Binghamton, N.Y.), 10% human AB serum and 10%dimethylsulfoxide (Sigma) for storage in liquid nitrogen.

For culturing, the cyropreserved liver stromal cells were thawed andcounted. The cultures were seeded at about 1.62×10⁶ cells/cm² incomplete medium (DMEM low glucose containing 4 mM L-glutamine, 10% FBS,and 1% Penicillin/Streptomycin). The LSCs were cultured and used inexperiments discussed more fully below. Methods known in the art wereused to characterize the phenotype of LSCs with respect to BMSCs andfibroblast cell lines purchased from American Type Culture Collection(ATCC) as follows:

Flow Cytometry Analysis

Briefly, cells were washed in PBS containing 5% FBS and blocked withimmunoglobulin prior to staining with fluorochrome tagged antibodies.Background staining was determined by incubating cells withisotype-matched fluorochrome-labeled immunoglobulins. About fiftythousand events (cells) were used for analysis of the LSCs on a BectonDickinson FACSCaliber flow cytometer using Cell Quest acquisitionsoftware. Results from the flow analysis are summarized in Table 1. A“NEG” designation in Table 1 indicates less than 0.01% positivestaining. It was observed that the surface marker, CD14, distinguishedLSCs from BMSCs and the fibroblast cell lines tested. In addition, CD133was observed to be a marker which is present in lower concentration onLSCs relative to BMSCs, and therefore may be used as a marker todistinguish LSCs from BMSCs.

TABLE 1 Surface Antigen Profile (% Positive Cells) Fibroblast SourceEMSC Cluster Cluster Connective Liver 005 006 008 005 009 ofDistribution Fetal Lung Skin Spleen Tissue HV-027 HV-029 (CE) (CE) (CE)(CW) (CW) Differentiation Passage# P15 P8 P6 P7 P10 P3 P3 P3 P3 P4 P3 P3CD3 (T cell) 1.9 1.2 0.7 8.7 2.8 0.1 0.95 2.1 3.8 3.4 1.8 2.6 CD11aLFA-1 NEG NEG NEG NEG NEG 0.55 NEG 1.3 NEG NEG NEG NEG alpha CD13 APN91.6 93.3 98 57.2 82.1 94.2 95.4 86.7 81.5 91.8 93.7 96.5 CD14 LPSr NEGNEG NEG NEG NEG 13.8 6.7 NEG NEG NEG NEG NEG CD19 PanB NEG NEG NEG NEGNEG NEG NEG NEG NEG NEG NEG NEG cell CD29 β-1 83.1 74.6 92 90.4 70.594.5 93.6 80.1 67.6 86.7 84.8 89.3 Integrin CD31 PECAM-1 0.8 0.5 0.5 0.80.1 0.9 0.6 2 0.5 1.3 0.9 0.1 CD34 Hemet. NEG 0.1 NEG 2.9 1 0.5 6.4 NEGNEG 0.5 NEG 0.3 Stem cell CD40 Co- NEG NEG NEG NEG NEG NEG NEG NEG NEG1.9 NEG NEG stimulation CD44 HCAM ND 91.2 94.9 89.3 67.9 96.3 95.4 91.590.3 86.1 93.1 96.1 CD45 Pan NEG NEG NEG NEG NEG NEG 0.1 NEG NEG NEG NEGNEG Leukooyte CD54 ICAM-1 76.2 19.3 46 80.9 73.6 94 85.2 55.8 47 17.935.7 49.8 CD80 B7-1 4.7 1.9 1.6 7.6 3.9 0.3 1.8 2.8 3.9 6 2.1 2.5 CD86B7-2 NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG CD90 Thy-1 92.485.7 97.7 93.3 70 95.7 95.5 87.5 77 93.1 90.4 92.4 CD105 Endoglin 26.831.5 67.1 80.7 42.8 89.5 90 62.5 56.5 58.5 64.7 43.5 CD119 INF-γ-r 3.7 21.1 0.5 0.5 5.2 5.2 22.3 0.8 NEG 3.8 1.3 CD120a TNFR1 NEG NEG NEG NEGNEG NEG NEG NEG NEG NEG NEG NEG CD123 IL-3-r NEG NEG 2.7 NEG 0.8 2.8 1.2NEG 0.2 0.3 3.3 NEG CD132 Common NEG NEG NEG NEG 4.2 NEG 5.8 NEG NEG NEGNEG NEG γchain CD133 AC 133 11.2 5 4.5 38.6 18.3 4 7.6 20.2 17.9 27.422.7 35.5 CD212 IL-12-r NEG NEG NEG NEG 1.6 NEG 1.2 NEG NEG NEG NEG NEGMHC Class I 93.5 70.4 96.5 91.8 90.9 96.8 92.9 88.5 76.1 93.5 89.4 92.6MHC Class II NEG NEG NEG NEG NEG 0.1 0.2 NEG NEG 0.1 NEG NEG

Differentiation Assay for Liver Derived Stromal Cells

Differentiation assays were performed on human BMSCs and human LSCs. Theassays tested the adipogenic and/or osteogenic differentiation potentialof the cells.

Adipogenic Assays

Cells were plated at 2×10⁵ cells/well in growth medium. At confluence,adipogenic differentiation was performed in three cycles ofinduction/maintenance medium. Maintenance media was DMEM supplementedwith 10% FBS, 10 μg/mL insulin, and 1 X antibiotic/antimycotic.Induction (differentiation) media was maintenance medium supplementedwith 10⁻⁶ M dexamethasone, 0.5 mM methyl isobutyl xanthine and 200 uMindomethacin. Each cycle encompassed a three day culture with adipogenicinduction media followed by a one day culture with adipogenicmaintenance medium. After this, the cells were washed with PBS, fixedwith 10% formalin, stained with Oil Red O, and counter-stained withhematoxylin to assess adipogenic differentiation.

Osteogenic Assays

Cells were plated at 2×10⁵ cells/well either in growth medium ordifferentiation media (DMEM containing 10% FBS, dexamethasone,beta-glycerophosphate and ascorbic acid-2-phosphate). Media for bothcontrol and differentiation wells were changed every 3-4 days for threeweeks. After this, the cells were washed with PBS and fixed with 10%formalin. Osteogenic differentiation was measured by assessing alkalinephosphatase activity and using von Kossa stain for mineral deposits.

The results of the differentiation assays are depicted in Table 2.

TABLE 2 Alk Phos Von Kossa Stromal Cells Adipogenic Results ResultsResults BMSCs (+control) +for lipid formation Strongly Strongly PositivePositive LSC #1 (HV-027) No Evidence of Lipid Weakly Negative Vacuoleformation Positive LSC #2 (HV-029) No Evidence of Lipid NegativeNegative Vacuole formation

It was observed that BMSCs differentiated into fat cells (adipocytes)and bone producing cells (osteoblasts) whereas LSCs did notdifferentiate into either lineage.

Example 2 Immunogenicity of Liver Stromal Cells

The experiments presented herein demonstrated that LSCs expressed novelimmunological characteristics in vitro. For example, LSCs were found tobe non-immunogenic when mixed with allogeneic T cells, as it wasobserved that LSCs when contacted with allogeneic T cells did not inducethe T cells to proliferate as compared with the amount of proliferationof T cells when contacted with allogeneic PBMCs. Also, it was discoveredthat LSCs were immunosuppressive for alloreactive T cell responses.Similar immunological characteristics have been described formesenchymal stem cells (MSCs) (Di Nicola et al., 2002 Blood 99:3838; Tseet al. 2003 Transplantation 75:389; Le Blanc et al. 2003 Scand. J.Immunol. 57:11), but not for the majority of fibroblast populationsderived from different tissue sources.

The materials and methods used in the experiments presented in thisExample are now described.

MLR Assays

The immunogenicity of LSCs were evaluated by mixed lymphocyte reaction(MLR) using T cells as responding cells and allogeneic PBMCs asstimulator cells. T cells were purified from leukopheresis packs(AllCells, LLC, Berkeley, Calif.; Poietics, Rockville, Md.) by negativeselection using mouse monoclonal antibodies (Serotec, Raleigh, N.C.)specific for monocytes (CD 14), B cells (CD 19), MHC class II, and NKcells (CD56) to label non-T cells for removal using magnetic beadscoated with monoclonal anti-mouse IgG antibody (Dynal Biotech, Inc.,Lake Success, N.Y.). The remaining population of cells after depletionwas typically greater than 85% CD3 positive by flow cytometry analysis.T cells were suspended in culture medium: Iscove's Modified Dulbecco'sMedium (IMDM) supplemented with sodium pyruvate, non-essential aminoacids, 2-mercaptoethanol, antibiotic/antimycotic, and 5% human AB serum(all supplements were obtained from Gibco, Carlsbad, Calif. except humanAB serum which was obtained from PelFreez, Brown Deer, Wis.). T cellswere seeded into microtiter wells (2×10⁵/well) in 96-well lowevaporation flat-bottom plates (BD Falcon, Franklin Lakes, N.J.) withallogeneic stimulator cells. Stimulator cells were irradiated with 5000rads gamma irradiation using a cesium source (Isomedix Gammator B,Parsippany, N.Y.) prior to being plated at the numbers necessary for theexperiment (usually titrated decrementally from a high dose of 5×10⁴cells/well). Cultures were set up in triplicate wells per treatment. Tcell proliferation to alloantigens was determined by pulsing thecultures with 3H-thymidine on the 6th day of culture. The cells wereharvested onto filtermats 16 hours later using a 96 well cell harvester(Skatron, Molecular Devices Corp, Sunnyvale, Calif.), and the cells onthe filtermats were counted using a Microbeta scintillation counter(Perkin Elmer, Turku, Finland).

Immunogenicity Experiments

The one-way MLR assay was used to evaluate T cell proliferation toallogeneic LSCs. Briefly, T cells (2×10⁵/well) were cultured withirradiated (5000 rads gamma radiation) allogeneic LSCs, autologousPBMCs, or allogeneic PBMCs (30,000 cells/well) in 96 well microtiterculture plates. The LSCs were obtained from two different donorsdesignated 027 and 029. T cells were purified from PBMCs obtained fromfour different donors designated 002, 004, 005, and 006. T cellenrichment was achieved by negative selection, using magnetic beads(Dynal, Inc) to remove non-T cells. Mouse monoclonal antibodies (mAbs)specific for macrophages/monocytes/dendritic cells (anti-CD 14), B cells(anti-CD 19), NK cells (anti-CD56), and MHC class II antigens (anti-DR)were used to label these cells. Magnetic particles coated with goatanti-mouse IgG antibody were used to pull the cells out in a magneticfield. The resulting cell population was typically greater than 90% Tcells by flow cytometry analysis using fluoresceinated anti-CD3 mAb todetect T cells. Cell culture medium used was Iscove's ModifiedDulbecco's Medium (IMDM) supplemented with 5% human AB serum,non-essential amino acids, sodium pyruvate, pen-strep/fungizone, and2-mercaptoethanol. The cultures were incubated in a humidifiedatmosphere of 5% CO₂ at 37° C. for 6 days, pulsed with 3H-thymidine (1μCi/well) for 16 hrs, and the cells were harvested on day 7 using anautomated 96 well cell harvester. The incorporated radioactivity wasdetermined by scintillation counting and the results are reported ascounts per minute (cpm).

In order for a test cell to be deemed immunogenic, three requirementsmust be met:

-   -   1) the test cell must induce a T cell proliferative response        that is at least 750 cpm above the autologous response;    -   2) the Stimulation Index (T+test cell cpm/T+autologous cell cpm)        must be greater than or equal to 3.0; and    -   3) there must be a statistically significant difference between        T +autologous PBMCs and T+test cells (P<0.05, Student's t test).

To rule out genetic factors (i.e., MEW similarities) as for the cause ofnon-responsiveness, each test cell was cultured with at least 2(preferably 3) different T cell donors. If any of the donors produced apositive response as described above, the test population was consideredimmunogenic. Using these criteria, it was concluded that fibroblastsderived from skin, spleen, lung, connective tissue, and fetal materialare immunogenic.

The results of MLR experiments using four different T cell donors asresponders and LSCs as stimulators, demonstrate that LSCs are notimmunogenic (FIG. 1). It was observed that the T cell response againstLSCs derived from the two different donors was similar to the responseagainst autologous PBMCs. It was also observed that the T cell responseagainst LSCs was significantly less than the response to allogeneicPBMCs. LSCs did not stimulate a significant response from any of thefour T cell donors as defined by the three criteria discussed above.

Suppression Experiments

LSCs were added to one-way MLR assays to determine whether they couldsuppress alloreactive T cell proliferation. Briefly, LSCs from twodifferent donors (027 and 029) were irradiated (5000R) and added toone-way MLR cultures comprising purified T cells (responder cells) andirradiated allogeneic PBMCs (stimulator cells). Purification of T cellsand culture conditions for the MLR are described elsewhere herein.Fibroblasts derived from connective tissue, fetal tissue, lung, skin andspleen, were also added to MLR cultures to determine their relativesuppression properties. All of these primary cell lines were purchasedfrom American Type Culture Collection (ATCC). BMSCs were tested fortheir suppressive property as well. LSCs, fibroblasts, and BMSCs,respectively, were added to MLR cultures between different combinationof T cells and PBMCs at a dose of 30,000 cells/well. Results are shownas percent suppression of the base MLR response to which nofibroblasts/stromal cells were added. Each bar represents the meansuppression of four different MLR cultures by each cell type (except forlung fibroblasts for which the mean was calculated from three differentMLR cultures). Suppression was determined by comparing the responsecontaining the test cells to the control MLR according to the followingformula:

Percent Suppression=[1−(cpm of MLR culture containing test cells/cpmcontrol MLR culture)]×100.

The results demonstrated that LSCs from both donors suppressed the MLRresponse to a greater extent than any other cell type that was evaluated(FIG. 2). The results shown are the averages of four experiments, eachexperiment using a different combination of T cells and PBMCs for theMLR. Connective tissue fibroblasts, skin fibroblasts, and bone marrowstromal cells were also observed to suppress the MLR response, whereasfetal fibroblasts, lung fibroblasts, and spleen fibroblasts enhanced theMLR response (negative ‘Percent Suppression’ indicates that the addedtest cell population enhanced the MLR response over control levels).

Example 3 Liver Stromal Cells in Transplantation

Given that LSCs suppress alloreactive T cell responses, as discussedelsewhere herein, LSCs can be co-transplanted with immunogenic cells,tissues, or organs to prevent immune rejection of the transplantedmaterial. An advantage to this approach over those currently used in theart is that the cells can be used to establish restricted areas ofimmune privilege in the area of transplant without generalizedsuppression of the immune system which can be detrimental to the host.

The immunosuppressive properties of LSCs can be exploited to enhance thesurvival of transplanted cells, tissues, or organs. Without being boundto any particular theory, it is believed that LSCs delivered to atissue/organ would create a localized area of immunosuppression orimmune privilege which would aid in engraftment of cells, tissues, ororgans. Although immune tolerance may be induced under these conditions,it is not required for the successful use of LSCs for the reductionand/or elimination of an immune response of effector cells against analloantigen in a transplant recipient. It is preferred that there is along-term engraftment of a sufficient number of LSCs at the desiredsite, wherein the LSCs maintain their suppressive phenotype.

The materials and methods used in the experiments presented in thisExample are now described.

Isolation of LSCs

As discussed elsewhere herein, LSCs were isolated from adult cadaverichuman livers designated Hu027 and Hu029. Briefly, the liver was perfusedvia portal vein and hepatic artery and the cells were passed through1000, 500, 250 and 150 μm filters. Viable cells were fractionated under500×g with COBE cell processor (Gambro BCT, Lakewood, Colo.) using2-step (9% and 17%; Hu027 and Hu029 cells) or 12.5% (H0107 cells)OptiPrep gradients (Axis-Shield PoC AS, Oslo, Norway).

Isolation and Culture of Human Fetal Neural Stem Cells

Human fetal brain tissue can be purchased from Advanced BioscienceResources (Alameda, Calif.). The tissue is washed with PBS/antibioticsand the meninges are removed prior to using the desired brain tissue.The remaining tissue is teased apart with a pair of forceps and furtherdissociated by trituration with a Pasteur pipette. Cells are thenpelleted by centrifugation at 1000 rpm for 5 minutes at roomtemperature. The cell pellet is resuspended in 10 ml of NSC growthmedium (DMEM/F 12, 8 mM glucose, glutamine, 20 mM sodium bicarbonate, 15mM HEPES, 8 μg/ml Heparin, N2 supplement, 10 ng/ml bFGF, 20 ng/ml EGF).The cells are plated on a coated (15 μg/ml Polyornithine overnightfollowed by 10 μg/ml human Fibronectin for greater than 4 h) T-25 cm²flask with vented cap and grown in a 5% CO₂ incubator at 37° C. Cellsgrown with Leukemia Inhibitory Factor (LIF) are plated in the completegrowth medium with 10 ng/ml LIF after growing them initially (1-2passages) in the presence of bFGF and EGF alone. Cultures are fed everyother day by replacing 50% of the medium with fresh complete growthmedium. Cells are passaged by trypsinization with 0.05% Trypsin-EDTA inPBS for 2-3 minutes followed by addition of soybean trypsin inhibitor toinactivate the trypsin. The cells are pelleted at 1200 rpm for 5 minutesat room temperature, resuspended in growth medium, and plated at1.0-1.25×10⁵ cells/cm² on coated flasks. Cells are cryopreserved in 10%DMSO+90% complete growth medium.

Human MLR Assays

The immunogenicity of stem cells can be evaluated by mixed lymphocytereaction (MLR) using T cells as responding cells and allogeneic PBMCs,NSCs, or LSCs as stimulator cells. If the stimulator cells areimmunogenic to the T cells, the stimulator cells will activate the Tcells and induce the T cells to proliferate. T cells used in theseexperiments are purified from leukopheresis packs as described elsewhereherein.

Rat MLR Assays

These assays are set up in similar fashion to the human MLRs. Briefly,responder cells are produced by harvesting cervical plus mesentericlymph node cells (LNCs). The responder cells are dissociated into asingle-cell suspension by grinding them with a syringe plunger against acell strainer (BD Falcon) in a 6-well plate. The responder cells aresuspended in culture medium similar to human MLR medium with theexception that the serum is 10% FBS (HyClone, Logan, UT). LNCs areseeded into microtiter wells (4×10⁵/well) with allogeneic stimulatorcells at the numbers necessary for the experiment. Stimulator cells areirradiated (5000 rads) prior to being plated. Cultures are set up usingtriplicate wells per treatment. T cell proliferation to alloantigens areassessed as described elsewhere herein.

Human Alu PCR Assay

The number of human NSCs in rat livers can be quantified using anintra-Alu element-based PCR assay described by Walker et al. (2003,Analytical Biochem. 315:122-128). The naturally occurring repetitive Alusequence present in human DNA allows greater detection sensitivity oversingle copy sequences/genes. Thus, genomic DNA of human origin isquantified via RealTime PCR for the human-specific Alu repeat sequence.The primers employed for this assay amplify an intra-Alu core repeatsequence of ˜200 bp within the Yb8 Alu subfamily. Use of these primerswas reported by Walker et al. (2003, Analytical Biochem. 315:122-128) toallow human DNA specific detection to at least 10 pg (˜1 cellequivalent) in 2 ng of mixed species sample DNA. Genomic DNA is isolatedfrom snap-frozen rat livers using the Puregene DNA Isolation kit (GentraSystems). Human DNA is quantitated by comparison with an Alu-specificDNA standard curve generated from populations of rat cells spiked (in10-fold increments) with known numbers of human cells.

Co-Transplantation of LSCs with NSCs

NSCs were chosen for the following experiments because NSCs represent anexample of a stem cell that has significant clinical applications.However, it is envisioned that any cell, tissue or organ can be used inthe following experiments. The following experiments address the role ofLSCs in suppressing an immune response against NSCs transplanted into arecipient.

NSCs Express MHC Class I Antigens

NSCs were prepared from human fetal tissue using methods well known inthe art and were cultured for about 13 passages. The cells wereevaluated for immunologically relevant cell membrane molecules usingflow cytometry. It was observed that the population of NSCs did notexpress hematopoietic markers (CD45, CD14, CD34), costimulatorymolecules (CD80, CD86), or MHC class II molecules. However, the NSCs didexpress the stem cell marker, CD133, as well as MHC class I antigens.The expression of class I molecules usually indicates that the cellswould be recognized by alloreactive T cells and would be rejected iftransplanted into an allogeneic recipient. Based on the disclosurepresented herein, LSCs are an exception to this dogma because it wasdemonstrated that LSCs are not immunogenic to T cells in MLRs.

NSCs Stimulate Proliferation of Allogeneic T Cells

The immunogenicity of NSCs was evaluated by one-way mixed lymphocytereaction (MLR) using T cells as responding cells and irradiated NSCs asstimulator cells. The MLR, which measures the T cell proliferativeresponse to alloantigens, is predictive for survival of allogeneic cellsin vivo. NSCs were prepared from human fetal tissue and cultured forabout 13 passages. Starting with a high dose of about 5×10⁴ cells/well,the cells were titrated down in 3-fold decrements as stimulator cells.Purified T cells (2×10⁵ cells/well) from an unrelated donor wereprepared as responder cells. Autologous PBMCs were used as controlstimulator cells. As shown in FIG. 3, NSCs stimulated a significantdegree of T cell proliferation compared to autologous PBMCs, which didnot stimulated a significant amount of T cell proliferation even at thehighest cell dose (P<0.05, Student's t test). These results demonstratethat allogeneic NSCs are recognized by T cells and induce a functionalimmune response.

The ability of LSCs to protect NSCs from rejection in vivo can beexamined using a xenogeneic model or an allogeneic model. Although theclinical model can involve xenogeneic transplantation of cells as wellas an allogeneic model, a xenogeneic model is described herein to serveas proof of principle. The xenogeneic model encompasses the followingcriteria:

-   -   1) human NSCs as the donor cell;    -   2) rat LSCs because human LSCs have been shown to be suppressive        for alloreactive T cell responses; and    -   3) the liver was chosen as the site of implantation due to the        ease of administration of cells through the portal vein, the        accessibility of the injected cells to the immune system, and        the ability to recover injected cells. Without wishing to be        bound to any particular theory, LSCs injected intraportally may        become lodged in the liver. Further, since NSCs are        approximately the same size as LSCs, NSCs may also become        trapped in the liver after portal delivery. The use of human        NSCs as the only human cell in this model enables the assessment        of the engraftment using a PCR technique specific for human Alu        DNA sequences. The model described herein also, can be used to        determine whether the NSCs and LSCs induce a T cell response in        the lymph nodes of recipient animals by using these cells in        one-way MLR assays.        Suppression of Xenogeneic Rat versus Human MLR by Rat LSCs

Xenogeneic MLRs can be set up between rat and human cells to evaluatewhether rat LSCs can suppress this response. Rat LSCs allogeneic to therecipient are used in these experiments, but LSCs from any source can beused to suppress MLR responses. For example, rat LSCs autologous to therecipient can also be used.

Fisher rat lymph nodes (LNs) are harvested, dissociated into asingle-cell suspension, and plated in microtiter wells (4×10⁵/well) asresponder cells in the MLR. Human NSCs and allogeneic PBMCs areirradiated and plated at 1×10⁵ cells per well as stimulator cells. LSCsand fibroblast from ACI strain rat are titrated into the MLRs at a highdose of 5×10⁴ cells/well and 2-fold decremental doses down to 3,125cells/well. Culture conditions for the rat MLR are as describedelsewhere herein. Suppression is determined by comparing the control MLR(no LSCs) to MLRs containing fibroblasts or LSCs. Statistical evaluationare performed using the Student's t test.

Determine Survival of Transplanted NSCs

Experiments were designed to assess the survival of NSCs in vivo afteradministration with control fibroblasts or LSCs. 1:1 ratio of LSCs toNSCs can be used which should be adequate for suppression in vivo inview of a 1:3 ratio of LSCs to stimulator PBMCs being sufficient tosuppress the MLR in vitro. Further, it is believed that PBMCs arestronger stimulators of T cells than NSCs, and therefore warranting the1:1 ratio of LSCs to NSCs.

Human NSCs (5×10⁶ cells) are mixed with ACI strain rat dermalfibroblasts (5×10⁶ cells) and are injected intraportally in a volume of200 μl into each of 25 Fisher rats. Dermal fibroblasts are produced fromskin samples obtained from ACI rats and expanded using similar methodsfor expanding LSCs as described elsewhere herein. Another group of 25Fisher rats are injected intraportally with human NSCs (5×10⁶ cells/rat)mixed with an equal number of ACI strain rat LSCs (5×10⁶ cells/rat).Five rats from each group are sacrificed on days 1, 7, 14, 21, and 28after injection. The livers are removed, snap frozen, and subject to theAlu PCR assay as described elsewhere herein in order to assess theengraftment of the human NSCs.

It is believed that LSCs can mediate localized suppression in vivo, andextend the survival of xenogeneic cells in the liver. Thus, a greaternumbers of human NSCs from rats that were given LSCs are recovered thanfrom rats that received NSCs with non-suppressive fibroblasts. Thegreatest difference between these two groups would be expected to occurafter 1-2 weeks, when the immune response is activated to the xenogeneicNSCs. A PCR assay can be used to detect human NSCs that have survivedthat transplantation by measuring the human-specific Alu repeatsequence.

Determine T Cell Priming to Injected Cells in Recipient Rats

Experiments are designed to determine whether human NSCs co-transplantedwith rat fibroblasts, or with rat LSCs primed reactive T cells inperipheral lymph nodes of recipient rats. A one-way MLR assay can beused to evaluate such priming.

Cervical and mesenteric lymph nodes (LNs) are removed from the twogroups of 5 rats each (human NSCs+rat fibroblasts vs human NSCs+ratLSCs) which are sacrificed at the final time point, one month afterinjection. The LNs from the rats are dissociated into a single-cellsuspension and used as responder cells in MLR assays with irradiateddonor human NSCs; rat fibroblasts and rat LSCs are used as stimulatorcells (5×10⁴ cells/well). Control groups used in these experiments canbe irradiated syngeneic Fisher strain spleen cells as stimulators(background), LNCs cultured in medium alone, and irradiated stimulatorcells cultured in medium alone. The mean response to each stimulatorpopulation are compared to background responses to syngeneic spleencells.

Not wishing to be bound to any particular theory, in the event thatxenogeneic human NSCs primed recipient rats, T cells from recipient ratsshould give a secondary response in the MLR assay to human NSCs asstimulator cells. In contrast, if LSCs administered to the recipient ofthe transplant prevented an immune response to the human NSCs followingco-transplantation of the LSCs and human NSCs, recipient T cells shouldgive a primary MLR response. If the transplanted LSCs tolerizedrecipient T cells to NSCs following co-transplantation of LSCs withNSCs, they should give reduced responses in an MLR.

Example 4 Co-Transplantation of LSCS with Islet Cells

LSCs can be used in co-transplantation with allogeneic islet cells forthe treatment of diabetes. Allogeneic islets are introduced into arecipient by injecting islets with LSCs into the portal vein of therecipient which carries the cells to the liver, where the islets take upresidence and function to produce insulin in response to glucose. Whilenot wishing to be bound to any particular theory, co-transplantation ofLSCs with allogeneic islet cells may function to protect the islets fromrejection by the host without the use of immunosuppressive drugs. LSCsmay also survive for extended periods of time in the liver since it istheir tissue of origin.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodifications and variations of the present invention provided they comewithin the scope of the appended claims and their equivalents.

What is claimed is:
 1. A method of inhibiting an immune response in atransplant recipient comprising: administering to the transplantrecipient, isolated adult liver stromal cells (LSCs), wherein the LSCsare incapable of differentiating into fat cells or bone producing cells,in an amount effective to inhibit an immune response by effector cellsagainst an alloantigen to the effector cells, thereby inhibiting immuneresponse in the transplant recipient.
 2. The method of claim 1, whereinthe effector cells are T cells.
 3. The method of claim 2, wherein the Tcells are from a donor and the alloantigen is from the recipient.
 4. Themethod of claim 2, wherein the T cells are from the recipient and thealloantigen is from a donor.
 5. The method of claim 2, wherein the Tcells are present in a transplant.
 6. The method of claim 1, wherein thetransplant recipient receives a bone marrow transplant, a hematopoieticstem cell transplant or a neural stem cell transplant.
 7. The method ofclaim 1, wherein prior to the administering liver stromal cells to atransplant recipient, the liver stromal cells have been expanded inculture.
 8. The method of claim 1, wherein the effector cells are Tcells from a donor previously activated by contacting the T cells with acell or a tissue from the recipient prior to transplantation in order toactivate the T cells, and further wherein the immune response is thereactivation of the T cells.
 9. The method of claim 1, wherein the liverstromal cells are administered to the transplant recipient to treatrejection of the transplant by the recipient.
 10. The method of claim 1,wherein the effector cells are T cells from a donor previously activatedby contacting the T cells with a cell or a tissue from the recipientprior to transplantation in order to activate the T cells, and furtherwherein the immune response is the reactivation of the T cells.
 11. Themethod of claim 1, wherein the liver stromal cells are human liverstromal cells.
 12. The method of claim 1, further comprisingadministering to the recipient an immunosuppressive agent.
 13. Themethod of claim 1, wherein the transplant is a solid organ selected fromthe group consisting of heart, pancreas, kidney, lung and liver.
 14. Themethod of claim 1, wherein the liver stromal cells are administered tothe recipient prior to a transplant.
 15. The method of claim 1, whereinthe liver stromal stem cells are administered to the recipientconcurrently with a transplant.
 16. The method of claim 17, wherein theliver stromal cells are administered as part of a transplant.
 17. Themethod of claim 1, wherein the liver stromal cells are administered tothe recipient subsequent to the transplantation of a transplant.
 18. Themethod of claim 1, wherein the liver stromal cells are administeredintravenously to the recipient.
 19. The method of claim 1, wherein theeffector cells are cells of a recipient of a donor transplant.
 20. Themethod of claim 1, wherein the liver stromal cells are geneticallymodified.